Method for introducing an exogenous DNA by overcoming the restriction modification barrier of a target bacterium

Abstract

Disclosed is a method for introducing an exogenous DNA by overcoming the restriction modification barrier of the target bacterium. The method includes the steps of 1) co-expressing all DNA-methyltransferase-encoding genes in the genome of the target bacterium in E. coli in which the restriction modification system thereof has been deleted to obtain a recombinant bacterium A, 2) introducing an exogenous DNA molecule into the recombinant bacterium A for in vivo modification so as to obtain a methylation-modified exogenous DNA molecule, and 3) introducing the methylation-modified exogenous DNA molecule into the target bacterium.

What is claimed is: 
     
       1. A method for introducing a methylated exogenous DNA molecule into a target bacterium, the method comprising the steps of:
 a) providing an original  E. coli  strain having a deficient recA gene; 
 b) repairing the deficient recA gene and knocking out the dcm gene and the dam gene of the original  E. coli  strain to obtain a modified  E. coli  strain; 
 c) co-expressing in the modified  E. coli  strain all putative DNA-methyltransferase-encoding genes encoding functional DNA methyltransferases in the genome of the target bacterium to obtain a recombinant  E. coli  bacterium A which shows the same DNA methylation pattern as that of the target bacterium; 
 d) introducing an exogenous DNA molecule into said recombinant  E. coli  bacterium A for in vivo modification to obtain a methylated exogenous DNA molecule and extracting the methylated exogenous DNA molecule to obtain an extracted methylated exogenous DNA molecule; and 
 e) introducing said extracted methylated exogenous DNA molecule into said target bacterium, 
 wherein the target bacterium is not an  E. coli  strain. 
 
     
     
       2. The method according to  claim 1 , wherein step c) is accomplished by introducing a recombinant expression vector carrying all of the putative DNA-methyltransferase-encoding genes into said modified  E. coli  strain; and step d) is accomplished by the substeps of:
 A) introducing said exogenous DNA molecule into said recombinant  E. coli  bacterium A to obtain a recombinant  E. coli  bacterium B; 
 B) inducing and culturing said recombinant  E. coli  bacterium B to obtain an induced recombinant  E. coli  bacterium B; and 
 C) extracting the DNA of said induced recombinant  E. coli  bacterium B to obtain said extracted methylated exogenous DNA molecule. 
 
     
     
       3. The method according to  claim 2 , wherein in substep B), said inducing is inducing by temperature or by arabinose, IPTG, xylose, or rhamnose. 
     
     
       4. The method according to  claim 3 , wherein substep B) is accomplished by culturing the recombinant  E. coli  bacterium B in a liquid culture medium containing arabinose at a final concentration of 0.2% by mass, the culturing being performed at a temperature of 25° C.-37° C. for 3-24 hours. 
     
     
       5. The method according to  claim 1 , wherein said target bacterium is  Bacillus amyloliquefaciens  TA208,  Bacillus cereus  ATCC 10987, or  Nitrobacter hamburgensis  X14; and said modified  E. coli  strain is  Escherichia coli  EC135, deposited as strain CGMCC No. 5925. 
     
     
       6. The method according to  claim 1 , wherein said exogenous DNA molecule is an exogenous plasmid DNA molecule. 
     
     
       7. The method according to  claim 5 , wherein said all putative DNA-methyltransferase-encoding genes encoding functional DNA methyltransferases of said  Bacillus amyloliquefaciens  TA208 are encoded by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5;
 said all putative DNA-methyltransferase-encoding genes encoding functional DNA methyltransferases of said  Bacillus cereus  ATCC 10987 are encoded by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or 
 said all putative DNA-methyltransferase-encoding genes encoding functional DNA methyltransferases of said  Nitrobacter hamburgensis  X14 are encoded by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. 
 
     
     
       8. The method according to  claim 1 , further comprising determining said all putative DNA-methyltransferase-encoding genes encoding functional DNA methyltransferases in the genome of the target bacterium as follows:
 (a) determining putative genes encoding DNA methyltransferases in a target bacterium by homologous sequence alignment; 
 (b) introducing each putative gene encoding DNA methyltransferases into  E. coli ; and 
 (c) preparing genome DNAs of the aforementioned  E. coli  and detecting whether DNAs have been methylated. 
 
     
     
       9. The method according to  claim 4 , wherein the culturing is performed at a temperature of 30° C. for 12 hours. 
     
     
       10. The method according to  claim 1 , wherein said target bacterium is an  Eubacterium  or Archaebacterium containing a restriction modification system.